ATP-sensitive potassium channels (K_ATP) are involved in a diverse array of physiologic functions including protection of tissue against ischemic insult, regulation of vascular tone, and modulation of insulin secretion. To improve our understanding of the role of K_ATP in these processes, we used a gene-targeting strategy to generate mice with a disruption in the muscle-specific K_ATP regulatory subunit, SUR2. Insertional mutagenesis of the Sur2 locus generated homozygous null (Sur2^−/−) mice and heterozygote (Sur2^+/−) mice that are viable and phenotypically similar to their wild-type littermates to 6 weeks of age despite, respectively, half or no SUR2 mRNA expression or channel activity in skeletal muscle or heart. Sur2^−/− animals had lower fasting and fed serum glucose, exhibited improved glucose tolerance during a glucose tolerance test, and demonstrated a more rapid and severe hypoglycemia after administration of insulin. Enhanced glucose use was also observed during in vivo hyperinsulinemic euglycemic clamp studies during which Sur2^−/− mice required a greater glucose infusion rate to maintain a target blood glucose level. Enhanced insulin action was intrinsic to the skeletal muscle, as in vitro insulin-stimulated glucose transport was 1.5-fold greater in Sur2^−/− muscle than in wild type. Thus, membrane excitability and K_ATP activity, to our knowledge, seem to be new components of the insulin-stimulated glucose uptake mechanism, suggesting possible future therapeutic approaches for individuals suffering from diabetes mellitus.